Abstract
Abstract The kinetics of the reaction between protoporphyrin and apocytochrome c peroxidase have been studied by spectrophotometric and stopped-flow methods. The reaction is composed of two steps which can be described by the following scheme: [see PDF for equation] The rapid first reaction (I) is assumed to be the chemical binding of the porphyrin with apocytochrome c peroxidase. The rate constants of this reaction were k1 = 1.40 x 106 m-1 s-1, k2 = 1.1 s-1 at 20c. The slow second reaction (II) was pH dependent and was associated with the conformational change of the protein. The rate constants of the second reaction were k3 = 5.9 x 105 m-1 s-1, and k4 = 7.1 x 10-3 s-1. The effects of pH, protein concentration, and ionic strength on these rate constants were also studied. Since the hemin combines very rapidly and irreversibly with apocytochrome c peroxidase, it was concluded that the iron atom is essential to accelerate the rate of the conformational change and to stabilize the protein conformation of cytochrome c peroxidase.
Highlights
The porphyrin wasdissolvedin a Kinetic studies of the reaction between heme and apohemoprotein are of interest becauseit is an extremely fast minimum volume of 0.1 N NaOH and diluted to an appropriate concentration immediately before use
Reaction oj Apocytochrome c Peroxidase wifh Protoporphyrin-Various iron-free porphyrins combine with apocytochrome c peroxidase to form well defined 1:l complexes [7]
(7), the addition of protoporphyrin to apocytochrome c peroxidase rapidly produces an intermediat,e complex having an absorption peak at 384 nm, which is slowly converted to a protoporphyrin-protein complex with an absorpt.ion peak at 408 nm
Summary
The free protoporphyrin was prepared by acid hydrolysis in 6 N HCl [11]. The concentration of the protopornhvrin was-determined by ‘the use of the extinction coeffiiient ;,,a-=” 262. The porphyrin wasdissolvedin a Kinetic studies of the reaction between heme and apohemoprotein are of interest becauseit is an extremely fast minimum volume of 0.1 N NaOH and diluted to an appropriate concentration immediately before use. Apocytochrome c Peroxidase-Yeast cytochrome c peroxidase biological reaction and because it is related to the biosynthe- was kindly provided by Dr T. The reaction betweenhemeand apohorse- peroxidasewaspreparedfrom yeast cytochromec peroxidaseby radish peroxidase was examined by Theorell and Maehly in 1950 the acid butanone method [6].
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